Molecular, serologic and functional approaches were utilized for the characterization of a monoclonal antibody to a human melanoma-associated 250 Kd glycoprotein-proteoglycan complex. The rationale for these studies was that it is necessary to understand antibody-antigen systems in order to optimize the therapeutic and diagnostic usefulness of monoclonal antibodies. The 259 Kd MAA, previously thought to be melanoma-specific, was detected on certain brain tumors in addition to cerebral metastases of melanoma but not on normal brain tissues. The antibody appears to have considerable diagnostic potential for brain tumors as well as melanoma. An important finding for therapeutic use of the antibody was that reactivity of antibody 9.2.27 to fresh human melanoma cells was characterized by high antigen density compared to other human MAA and a low degree of heterogeneity. This was shown to be dependent on the epitope recognized by a monoclonal antibody; other antibodies to the 250 Kd MAA reacted less well and gave a higher degree of heterogeneity on tissue sections and single cell suspensions. Preliminary data indicate that the 250 Kd MAA is functionally related to Alpha2-macroglobulin and can bind proteases. These studies are continuing to pursue means of characterization of this melanoma-associated antigen with regard to tumor cell biology and therapeutic applications.